Cholesterol is a key element in HIV replication and is required for various steps of the viral life cycle. HIV assembly occurs in cholesterol-rich domains of the plasma membrane and cholesterol is critical for HIV assembly and infectivity. However, little is known about perturbations of intracellular cholesterol homeostasis caused by HIV. Our preliminary data indicate that HIV-1 infection impairs an important branch of intracellular cholesterol metabolism, cholesterol efflux. In this application, we propose to explore the mechanisms of HIV-1-mediated effects on intracellular cholesterol metabolism. These mechanisms may have implications for lipid-related diseases often associated with HIV-1 infection, as well as present a possible target for anti-HIV therapeutic approaches. The objective of the proposed research is to analyze the effects of HIV infection on intracellular cholesterol metabolism using in vitro and in vivo models. This analysis will be performed at the cellular and molecular levels by addressing the following Specific Aims: Specific Aim 1. To investigate the effect of HIV-1 infection on intracellular cholesterol metabolism in vitro. We will identify pathways of intracellular cholesterol metabolism affected by HIV-1 and reveal the viral and cellular proteins responsible for this effect using in vitro models. Specific Aim 2. To investigate the effect of HIV-1 infection on intracellular cholesterol metabolism in vivo. The effect of HIV-1 infection on cholesterol efflux from macrophages will be investigated in vivo using mice injected with macrophages containing labelled cholesterol and transfected with HIV genes or infected with HIV. We are especially well prepared to successfully carry out this project as our team combines experience of Dr. D. Sviridov, a recognized expert in cholesterol transport and atherosclerosis, and Dr. M. Bukrinsky, an experienced HIV molecular virologist. This exploratory proposal is fully consistent with the R21 mechanism, which will provide short-duration support for initial studies to test our innovative hypothesis. These studies are expected to yield sufficient information upon which to base a well-planned and rigorous series of further investigations. [unreadable] [unreadable]